Multispectral illumination device

ABSTRACT

An illumination device includes at least four semiconductor radiation sources ( 18 ) for emitting optical radiation in respectively different emission wavelength ranges. At least one color splitter ( 22.1, 22.2, 22.3 ), which is reflective for optical radiation of the respective semiconductor radiation source ( 18 ), is assigned to each of at least three of the semiconductor radiation sources ( 18 ). The semiconductor radiation sources ( 18 ) and the color splitters ( 22.1, 22.2, 22.3 ) are arranged such that the optical radiation, which is emitted in each case from each of the semiconductor radiation sources ( 18 ), is coupled into a common illumination beam path section ( 24 ). In each case, one collimating unit ( 20.1, 20.2, 20.3, 20.4 ), which collimates the optical radiation emitted by the respective semiconductor radiation source ( 18 ), is arranged in the beam path sections from the semiconductor radiation sources ( 18 ) to the color splitters ( 22.1, 22.2, 22.3 ).

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation application of U.S. patent application Ser. No.12/083,980, filed Apr. 23, 2008 now U.S. Pat. No. 8,097,865, which isthe national stage of PCT/EP 2006/010745, filed Nov. 9, 2006,designating the United States and claiming priority from German patentapplication no. 10 2005 054 184.4, filed Nov. 14, 2005, the entirecontents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a multispectral illuminatingarrangement as well as optical units, especially, imaging units,examining units, observing units and projection units having such anilluminating arrangement.

BACKGROUND OF THE INVENTION

An important area of use of illuminating units is imaging units andexamining units which are provided for the purpose of generating imagesof an object or a specimen to be examined. Typical examples for suchimaging units are microscopes and especially microscopes having a widefield optic which images a pregiven region of the specimen, which is tobe imaged or examined, and not only a small point-shaped region of thespecimen, onto an image plane. The fluorescence examination has aspecial role for the optical examination of specimens. Here, thespecimen is irradiated with excitation radiation having a suitableexcitation spectrum which is selected in dependence upon one or severalfluorescence colorants. If these fluorescence colorants are in thespecimen, then they interact with the excitation radiation and emitfluorescence radiation which is characteristic for the fluorescencecolorant. In this way, a detection of the fluorescence colorants in aspecimen is possible. Not only can the presence of the fluorescencecolorants be determined but also their concentration can be deter mined.

An important area of application of fluorescence examinations ismolecular biology. Here, fluorescence colorants are used whichspecifically bond to pregiven substances in a biological specimen andcan then be detected in the bonded state. A conclusion as to thepresence and the concentration of the pregiven substance in the specimenis possible. For such examinations, so-called fluorescence readers areused which are designed for examining biochips having several regions(mostly even very many regions) delimited from each other withrespectively different chemical compositions.

In lieu of fluorescence colorants, also nanoparticles, for example,quantum dots, can be used which fluoresce at at least one wavelength.

Two basic problems occur in such fluorescence examinations. First, thefluorescence intensities are often very low so that influences of otheroptical radiation in adjacent wavelength ranges or other origins on thedetection of fluorescence radiation should be reduced as much aspossible in order to obtain a favorable signal-to-noise ratio. For thispurpose, illuminating devices are typically used which output excitationradiation with an only very narrow excitation spectrum. On the otherhand, emission filters are placed in the detection beam path for thefluorescence radiation from the specimen to a detector. The emissionfilters preferably pass only radiation in the wavelength range of thefluorescence radiation.

Secondly, for a pregiven fluorescence colorant, the spectrum of theradiation with which the fluorescence of the colorant is excitable aswell as also the fluorescence spectrum are specific so that differentcombinations of excitation radiation and emission filters are necessaryfor the detection of different fluorescence colorants.

Accordingly, it would be desirable for the rapid examination ofspecimens as to different fluorescence colorants to have an imaging andexamining arrangement which permits a change between the differentexcitation spectra and/or emission spectra.

For this purpose, a filter wheel having different emission filters can,for example, be used in lieu of a fixed emission filter in the detectorbeam path. Each one of the emission filters is provided for a pregivenfluorescence spectrum. A white light source can especially be used as anillumination source. This solution has, inter alia, the disadvantagethat, on the one hand, only a sequential examination of a specimen as toseveral fluorescence colorants can be carried out and, on the otherhand, mechanical parts must be moved for a change between theexaminations.

Additionally or alternatively, a multispectral illumination device canbe used with which sequentially and/or parallelly optical radiation canbe outputted in at least two different wavelength ranges. Opticalradiation in at least two different wavelength ranges is understood tobe radiation having an intensity with a pronounced maximum in thewavelength ranges. The intensity must not necessarily vanish in theregion between the two wavelength ranges.

In United States patent application publication US 2006/0187542, adevice for illuminating objects with light at different wavelengths isdescribed for microscopes, automatic microscopes and apparatus forfluorescence microscopic applications. This device includes LEP lightsources for object illumination Which are arranged in the illuminatingbeam path of the microscope or apparatus. For moving at least one of theluminescent diodes into an illuminating beam path, a mounting device,which is rotatable about a rotational axis, is provided and has holdersfor at least one of the luminescent diodes. The mounting device isadjustable via a drive unit so that the luminescent diode can be movedwith the centroid wavelength into the illuminating beam path with thecentroid wavelength being needed for the particular measurements and/orobservations.

This solution too is disadvantageous in that mechanically movable partsare needed and only a sequential examination is possible.

The use of mechanically movable parts has several disadvantages. Thus,the change from the detection of one fluorescent colorant to anothertakes a certain time which delays the examination of specimens.Furthermore, the assembly is complicated and subjected to wear. Finally,it is very complex to satisfy the required accuracy conditions for theadjustment of the filters or the light sources to the detection beampath or illumination beam path.

In U.S. Pat. No. 6,372,485 and United States patent applicationpublication US 2005/0224692 A1, illuminating devices having three LEDsare described. The radiation of these luminescent diodes is coupled intothe same illuminating beam path. The described devices have, however,the disadvantage that the power of the luminescent radiation in thedesired wavelength range is not very high compared to the radiationpower of the luminescent diodes. Furthermore, the not so compactconfiguration does not allow a high light-conductance value.Furthermore, with three LEDs, no adequate spectral range for theexcitation of fluorescence radiation can be covered.

SUMMARY OF THE INVENTION

The present invention is therefore based on the object of providing anilluminating device for outputting optical radiation having pregivenselectable spectra with the illuminating device being easily assembledand having only slight losses with respect to radiation power.

The object is solved with an illuminating device having at least foursemiconductor radiation sources for outputting optical radiation atrespectively different emission wavelength ranges. At least three of thesemiconductor radiation sources are each assigned at least one colorsplitter which is reflective for optical radiation of the correspondingsemiconductor radiation source. The semiconductor radiation sources andthe color splitters are so arranged that the optical radiation, which isemitted by each of the semiconductor radiation sources, is coupled intoa common illuminating beam path section. A collimation unit is mountedin each of the beam path sections from the semiconductor radiationsources to the color splitters. Each of the collimator units collimatesthe radiation outputted by the corresponding semiconductor radiationsource.

The illuminating device according to the invention therefore has nomovable parts by means of which, to switch over between wavelengthranges, one of the semiconductor radiation sources or a deflectionelement for optical radiation emitted by these radiation sources must bemoved. Rather, only at least four semiconductor sources are provided incombination with at least three corresponding color splitters in orderto selectively generate optical illuminating radiation having a pregivenspectrum.

The semiconductor radiation sources can be any desired semiconductorcomponent element emitting optical radiation. For example, laser diodesor superluminescence diodes can be used. Preferably, however, lightemitting diodes or luminescent diodes are used. Especially preferred arehigh power luminescent diodes which radiate optical radiation of highintensity. Preferably, high power color luminescence diodes are used forthe illumination source. In order to cover pregiven wavelength ranges,also luminescent diodes with applied luminescent substances for colorconversion can be used. These luminescent diodes output opticalradiation in a different wavelength range when excited by opticalradiation outputted by the semiconductor material of the luminescentdiodes.

The luminescent diodes can be especially those having a planar surfacefrom where the optical radiation emanates or luminescent diodes having atransparent dome for reducing refraction index discontinuities to thesemiconductor material. The transparent domes are arranged over thesurface.

The semiconductor radiation sources are configured for outputtingoptica1 radiation in respectively different emission wavelength ranges,that is, they exhibit respectively different emission wavelength rangesor emission spectra. This means that the characteristic wavelengths ofthe respective emission spectra (for example, intensity maxima of therespective emission spectra or centroid wavelengths of the respectiveemission spectra or their dominant wavelengths) are spaced from eachother, preferably by more than 50 nm. Specifically, the emission peaksof the semiconductor radiation sources preferably do not overlap atleast within the half wave width of the peaks, or only in the flanks ofthe emission peaks. The use of four semiconductor radiation sourcespermits a good covering of a given spectral range, especially of thetotal optical spectrum, especially of the visible spectrum as well asthe UV range and NIR range. Preferably, at least one of thesemiconductor radiation sources is, for this purpose, a UV radiationsource or an NIR radiation source having a characteristic emissionwavelength in the UV range or NIR range.

In order to make available a radiation power in a particular emissionwavelength range, which is adequate for a particular application, it isalso possible to use a corresponding field of like semiconductorradiation sources in lieu of only one semiconductor radiation source fora given emission wave region. For example, a field of like luminescentdiodes can be used.

The optical radiation which is emitted by each of the semiconductorradiation sources is coupled into a common illuminating beam pathsection and color splitters are used for this purpose. A color splitteris understood to be every optical element which is transmissive in apregiven wavelength range and is reflective in other pregiven wavelengthranges. In the illumination arrangement, respective color splitters areassigned to at least three of the at least four semiconductor radiationsources. For the optical radiation of the respective semiconductorradiation sources, the color splitters are either mostly (preferablyessentially) reflective or mostly (preferably essentially) transmissivein the region of the emission wavelength range, especially of thecharacteristic emission wavelength range. With a corresponding switchingin and switching out of the semiconductor radiation sources, opticalradiation corresponding to the emission wavelengths of the respectiveswitched in semiconductor radiation sources can be outputted asilluminating radiation along the common illuminating beam path section.In this way, a change of the semiconductor sources via mechanicalmovement in an illuminating beam path is not necessary and this hasseveral advantages. A rapid change is possible between differentemission wavelength ranges. Furthermore, the configuration of the deviceis very simple and of low wear.

Compared to conventional light sources such as arc lamps or halogenlamps, the use of semiconductor radiation sources, especiallyluminescent diodes, has furthermore the advantage that they have asignificantly longer service life. Furthermore, the thermal developmentby semiconductor radiation sources is significantly less during theiroperation than in arc lamps or halogen lamps. An active cooling of thesemiconductor radiation sources is therefore unnecessary and a passivecooling can be clearly simplified compared to the use of arc lamps orhalogen lamps.

A collimating device is mounted in each of the beam path sectionsbetween the corresponding ones of the semiconductor radiation sourcesand the color splitters. The collimating devices collect or moreprecisely essentially collimate the optical radiation of the respectivesemiconductor radiation sources. In this way, a very large portion,preferably more than 90%, of the optical radiation, which is emitted bythe semiconductor radiation sources, can be directed onto the colorsplitters so that, overall, a very high efficiency of the semiconductorradiation sources in the illuminating device can be achieved. Thecollimation furthermore has the advantage that the at leastquasi-parallel ray bundle, which arises with the collimation, permitsthe use of interference filters which permit an especially precisefiltering. Also, the function of the color splitters is, as a rule,better with the use of collimated radiation than with the use ofuncollimated radiation.

Overall, such an illumination device is generated which not onlyprovides a very simple and low wear configuration because of theomission of parts which have to be moved mechanically, but also outputsa very high illuminating radiation power referred to the radiation powerof the semiconductor radiation sources.

A possible application is the spectral separation in multicolorfluorescence. Here, it can be advantageously utilized that specificcolorants can hardly be excited and others, in contrast, can bemaximally excited via switching in and switching out individualsemiconductor radiation sources. Based on several such measurements, thequantity ratio of the colorants can be determined mathematically.

Furthermore, also a pixel shift can be avoided which often arisesbecause of a mechanical filter exchange. The radiation load on thespecimen can be minimized by the rapid switchability of thesemiconductor radiation sources. A mechanical shutter for theillumination is no longer needed.

The illumination source is suitable for all types of microscopes. Thefollowing, for example, belong to these types of microscopes: universalmicroscopes, optical readers (inter alia, biochip readers, titer-platereaders), stereo microscopes, surgical microscopes and ophthalmologicapparatus.

Preferably, the illuminating device includes a control unit by means ofwhich the semiconductor radiation sources can be switched on and offindependently of each other. This embodiment affords the advantage thata selection is easily possible between different emission wavelengthranges of the illuminating radiation outputted by the commonilluminating beam path section. The control unit can, for this purpose,include operator-controlled elements such as switches which are to beoperated by the user. Preferably, however, a control input is providedvia which the control unit can receive control signals in response towhich the control unit switches one or several of the semiconductorradiation sources on and off. The semiconductor radiation sources can beswitched on and off individually or also in combination with each other.This embodiment permits an especially rapid change between differentemission wavelength ranges and, with a combination of several emissionwavelength ranges, a simultaneous illumination with radiation indifferent emission wavelengths and therefore a simultaneous detection ofseveral fluorescence colorants in a specimen when the emissionwavelength ranges of the semiconductor radiation sources and the colorsplitters are correspondingly selected.

Here, it is especially advantageous that the control unit is soconfigured that the emission radiation powers of the semiconductorradiation sources can be adjusted independently of each other. Thisembodiment affords the advantage that the radiation powers of theindividual semiconductor radiation sources can be precisely adjusted incontrast to arc lamps and halogen lamps without a change of the colorspectrum. It is especially possible to so adjust the radiation powersthat the optical radiation, which is emitted by the semiconductorradiation sources into the common illuminating beam path section, hasrespectively the same emission radiation power.

Basically, different numbers of semiconductor radiation sources can beused. Preferably, the radiation device, however, has a total of five toeight semiconductor radiation sources having respective emissionwavelength ranges. Corresponding color splitters are assigned to atleast four to seven of these semiconductor radiation sources and theemission radiation of the semiconductor radiation sources is coupledinto the common illuminating beam path section via the respective colorsplitters. In this way, an especially good coverage of a given spectralrange is possible, especially of the total optical spectrum.

The beam paths from the semiconductor radiation sources to the lastcolor splitter in the illuminating beam path of the illuminating deviceahead of the common illuminating beam path section can run in differentways. For an especially compact form of the illuminating device, thebeam paths from the semiconductor radiation sources up to directlybehind the last color splitter ahead of the common illuminating beampath section form preferably a binary tree. As in graph theory, a binarytree is understood to be a tree having a root and, extending from theroot, branches, several knots connected to the root via the branchesand, at the free ends of the branches, which extend from the knots orroot, there are leaves wherein each knot is connected to at most twoother knots. Such a binary tree therefore has 2^(N) leaves for 2^(N)-1knots and these leaves are given by the semiconductor radiation sourcesin the illuminating device. N identifies a natural number greaterthan 1. The knots and the root correspond respectively to the colorsplitters while the semiconductor radiation sources define the leaves.The illuminating device therefore contains 2^(N) semiconductor radiationsources having respectively different emission wavelength ranges and atleast 2^(N)-1 corresponding color splitters. This embodiment affords theadvantage that the illuminating device can have an especially compactconfiguration.

The collimation units can be configured to be the same or respectivelydifferent. Especially, concentrators or holographic or diffractiveelements can be used which permit a collimation of the optical radiationoutputted by corresponding ones of the semiconductor radiation sources.Preferably, however, at least one of the collimating units includes anaspheric lens or an aspheric mirror. This embodiment of the collimatorunit affords the advantage that the aspheric configuration permits anespecially good bundling of the optical radiation outputted by therespective semiconductor radiation source and thereby radiation lossesvia less directed radiation of the optical radiation from thesemiconductor radiation sources can be reduced.

In order to make available the illuminating radiation with as narrow aspossible and sharply defined spectra, it is preferable that in at leastone radiation beam section from one of the semiconductor radiationsources to one of the color splitters, at least one bandpass filter ismounted preferably between the collimating unit which is mounted in thebeam path section and the color splitter. The bandpass filter functionsto mask long spectral extensions of the emissions spectra of thesemiconductor radiation sources and thereby possibly avoid disturbingbackground signals. This affords the advantage that a latermultispectral evaluation is facilitated, for example, for a fluorescenceexamination. The bandpass filter preferably has a spectral width of lessthan 100 nm.

Semiconductor radiation sources are up to now not available for anydesired emission wavelength ranges at economical cost. It is thereforepreferred that the illuminating device includes at least onesemiconductor radiation source having at least one luminescencesubstance for color conversion. A bandpass filter is mounted in the beampath of the semiconductor radiation source and is coupled likewise intothe common illuminating beam path section. The bandpass filter isdisposed preferably between a collimating unit assigned to thesemiconductor radiation source and the semiconductor radiation sourceitself. A color conversion via a luminescence substance is understood tobe that the luminescence substance converts radiation, which isoutputted from a radiation emitting layer of the semiconductor radiationsource, into radiation of the desired wavelength range via fluorescenceand/or phosphorescence, that is, radiation of another color. Thebandpass filter is so selected that the radiation of the semiconductorradiation source is spectrally limited to a suitable wavelength range.Preferably, that range lies in the region of 570 nm. This embodimentaffords the advantage that gaps in the wavelength range of the opticalspectrum, which is covered by the semiconductor radiation sources, canbe filled in that a corresponding bandpass filter is used.

Basically, any number of color splitters can be used which permit anin-coupling of optical radiation into respective emission wavelengthranges of the semiconductor radiation sources into the commonilluminating beam path section. Preferably, three color splitters aremounted in one and the same beam path section of the illuminating deviceand have filter edges at wavelengths which rise or fall along the beampath section. The semiconductor radiation sources whose emissionradiation passes through these color splitters then exhibit emissionwavelength ranges whose characteristic emission wavelength rises andfalls monotonically in the corresponding sequence. As color splittersespecially those can be used which have a spectral transmissioncapability in step form (that is, step filters) or band form. Thewavelengths at which the steps occur can then monotonically rise orfall. This selection of color splitters affords the advantage that asuccessive in-coupling of the optical radiation can take place withrespectively different emission wavelength ranges with only slightlosses.

Preferably, a homogenizing unit is mounted in the common illuminatingbeam path section downstream of the color splitters. The homogenizingunit functions to homogenize the intensity distribution of the opticalradiation, which is outputted by one or several of the semiconductorradiation sources, over the cross section of the resulting illuminatingbeam. The homogenization unit affords the advantage that a uniformillumination of the specimen is easily possible. As a homogenizing unit,especially transparent or hollow rods having reflecting side walls canbe used as can diffractive optical elements or scatter plate discs. Theuse of rods or hollow rods having reflective walls affords the advantagethat the losses therein are especially low.

Preferably, the light conductance values of the semiconductor radiationsources and the downstream optical elements of the illuminating deviceare adapted to each other for maximizing the energy flow of theradiation to be emitted. In this way, especially losses can be minimizedwhich are caused by the geometric arrangement of the semiconductorradiation sources and the optical elements so that illuminatingradiation having a high intensity relative to the radiation power of thesemiconductor component elements can be achieved.

The illuminating device does not necessarily have to be used to generateoptical radiation in only one of the emission wavelength ranges. Thus,in a preferred embodiment, the control unit is configured to drive atleast two of the semiconductor radiation sources to generate white lightof variable color temperature via additive color mixing of the opticalradiation emitted by the at least two semiconductor radiation sources.This embodiment affords the advantage that light of a pregiven colortemperature can easily be generated.

The illuminating device is suitable generally for illuminating purposeswhich require a rapid variation of the color spectrum. The subjectmatter of the invention is therefore especially also an opticalarrangement having an illuminating device according to the invention. Inaddition to the illuminating device, the optical arrangement includes atleast one further optical component which is mounted in the beam path ofthe radiation outputted by the illuminating device.

Especially, the illuminating device can preferably be used for digitalprojection systems. In this case, an especially wide color range iscovered. The subject matter of the invention is therefore also anoptical arrangement for projection of color images and/or films havingan illuminating device according to the invention. This can especiallyhave a projection optic arranged in the beam path of the radiationemitted by the illuminating device.

The illuminating device is especially preferred, however, for examiningspecimens. The subject matter of the present invention is therefore anoptical arrangement for examining a specimen, especially, a wide fieldmicroscope or a fluorescence reader having an illuminating deviceaccording to the invention. Such an examining arrangement ischaracterized in that it has only few or no movable parts caused by theconfiguration of the illuminating device and is therefore cost effectiveand simple to manufacture. Furthermore, the examining arrangement can bevery compactly configured.

Preferably, the arrangement is configured for carrying out fluorescenceexaminations on a specimen and, for this purpose, has at least onemultiband emission filter or multiband emission filter set in adetection beam path for fluorescence radiation emanating from thespecimen. In the following, and for the sake of simplicity, a multibandemission filter set is also identified as a multiband emission filter.The multiband emission filter can function especially as an emissionfilter for generated fluorescence radiation. Arrangements built up inthis manner for carrying out fluorescence examinations have significantadvantages compared to known fluorescence examining arrangementsespecially when the semiconductor radiation sources can also be drivensimultaneously. Accordingly, in a combined operation of at least two ofthe semiconductor radiation sources, simultaneous detection of severalfluorescence colorants in the specimen is possible which significantlyaccelerates the examination of specimens especially in sequentialexaminations. Furthermore, an exchange of the emission filters and animage offset occurring possibly as a consequence thereof can be easilyavoided via the switchover of two different semiconductor radiationsources. The image offset arises because of mechanical filter exchange.Furthermore, the radiation load on a specimen can be reduced during theexamination because of the rapid switchover between the differentsemiconductor radiation sources. The multiband emission filter ispreferably so configured that, in combination with a correspondingselection of the excitation spectra or emission spectra of theilluminating device, fluorescence examinations of at least two,preferably three to four, fluorescence substances of a specimen arepossible. For this purpose, especially the excitation spectra are to bematched to the fluorescence colorants.

To obtain an especially high signal-to-noise ratio, the device includespreferably a multiband excitation filter in the illuminating beam path.The excitation spectra can be very sharply defined because of thismultiband excitation filter. Furthermore, a multiband color splitter isprovided by means of which the illuminating radiation or excitationradiation, which is outputted by the illuminating device, can be coupledinto the illuminating beam path and/or imaging beam path of theexamining arrangement. This embodiment likewise affords the advantagethat an especially good signal-to-noise ratio is obtained because themultiband color splitter also operates as a filter. The bands arepreferably adapted to the excitation spectra and fluorescence spectra ofthe fluorescence colorants utilized.

In an especially preferred embodiment, the multiband emission filter iscontained in a filter cube which further has a multiband filter ormultiband filter set, which is arranged in the illuminating beam path ofthe examining device, and a multiband color splitter for deflecting theilluminating radiation of the illuminating device or deflectingfluorescence radiation emanating from the specimen. This affords theadvantage that a very compact configuration is obtained and an alignmentof the optical elements with respect to each other is simplified.

Preferably, in the fluorescence examining arrangement, at least one ofthe emission spectra of the illuminating device lies with itscharacteristic wavelength between two emission wavelength ranges of themultiband emission filter. This affords the advantage that via theexcitation radiation (that is, the emission radiation of thesemiconductor radiation sources of the illuminating device) littleoptical radiation can affect the detection of the fluorescenceradiation. Preferably, in the arrangement, the centroids of the emissionspectra of the illuminating device alternate with the emissionwavelength ranges of the emission filters. The characteristic emissionwavelength can be especially a wavelength wherein the emission spectrumhas a maximum or a centroid wavelength, which results as a medium valueof the emission wavelength weighted with the emission intensity, or thedominant wavelength.

Preferably, the device includes at least two multiband filters havingfilter edges which are shifted relative to each other by a distance ofbetween 10 nm and 90 nm. This embodiment affords the advantage that agreater number of different fluorescence colorants can be used forexamination.

It is especially advantageous when the examining arrangement includes areceptacle for at least two filter cubes by means of which one of thefilter cubes is movable into a detection beam path of the arrangement.This embodiment affords the advantage that a rapid exchange of thefilter cubes is possible which especially can greatly acceleratesequence examinations of specimens, especially, with biochip readers.The receptacle can be configured as a slider, wheel or turret. Themovement of the receptacle can be manual or the receptacle can be movedby an electric drive. A correspondingly greater number of semiconductorradiation sources with respectively different emission spectra is notnecessarily needed.

As already explained, the examining arrangement can preferably beconfigured as a microscope especially as a wide field microscope. Themicroscope can especially also be designed as an inverse microscope.

The subject matter of the invention is also a method for measuring theconcentration of a number M of pregiven fluorescence colorants in aspecimen wherein M measurements are carried out with a fluorescenceexamining arrangement, especially, a fluorescence examining arrangementaccording to the invention. For each of the measurements, another numberless or equal to M of emission wavelength ranges, which correspond tothe excitation spectra of the fluorescence colorants, are used forforming excitation radiation and the specimen is irradiated with theexcitation radiation and the resulting fluorescence radiation isdetected. The concentration of the fluorescence colorant is determinedfrom the detection results of the M measurements. M is a natural number.Compared to a method wherein M measurements are sequentially carried outfor only one fluorescence colorant in each case, this method affords theadvantage that possible excitations of fluorescence radiation by anotherthan the semiconductor radiation source, which is actually provided forthe excitation of given fluorescence colorants, can be mathematicallyeliminated. For the M measurements, especially a successively increasingor decreasing number of emission wavelength ranges or excitationwavelength ranges can be used.

Preferably, however, especially also the following include anilluminating device according to the invention: an optical arrangementfor examining the eye, especially a fundus camera, or an opticalarrangement for examining and/or viewing tissue, especially a surgicalmicroscope.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be explained in greater detail in the following withthe reference to the drawings.

FIG. 1 shows a schematic of a fluorescence examining arrangement in theform of a wide field fluorescence microscope according to a firstpreferred embodiment of the invention having an illuminating deviceaccording to a preferred embodiment of the invention;

FIG. 2 is a schematic representation of the emission spectra of thefollowing: the semiconductor radiation sources of the illuminatingdevice in FIG. 1, the transmission performance of the excitation bandfilters of the illuminating device in FIG. 1 and the transmissionperformance of the multiband color filters for the fluorescenceradiation of the examining arrangement of FIG. 1;

FIG. 3 is an illuminating device according to a second preferredembodiment of the invention;

FIG. 4 is a schematic representation of the emission spectra of thesemiconductor radiation sources and of the spectral transmissionperformance of the color splitters of the illuminating device of FIG. 3;

FIG. 5 shows an illuminating arrangement according to a third preferredembodiment of the invention;

FIG. 6 shows a schematic representation of the emission spectra of thesemiconductor radiation sources and of the spectral transmissionperformance of the color splitters of the illuminating device of FIG. 5;

FIG. 7 shows an illuminating device of a fourth preferred embodiment ofthe invention;

FIG. 8 is a schematic representation of the emission spectra of thesemiconductor radiation sources and of the spectral transmissionperformance of the color splitters of the illuminating device of FIG. 7;

FIG. 9 shows an illuminating device of a fifth preferred embodiment ofthe invention;

FIG. 10 shows a schematic representation of the emission spectra of thesemiconductor radiation sources and of the spectral transmissionperformance of the color splitters of the illuminating device of FIG. 9;

FIG. 11 shows an illuminating device according to a sixth preferredembodiment of the invention;

FIGS. 12 a, 12 b and 12 c show a schematic representation of thesemiconductor radiation sources and different embodiments of collimatordevices arranged ahead thereof for illuminating devices of additionalpreferred embodiments of the invention; and,

FIG. 13 shows a schematic representation of an examining arrangementaccording to a further preferred embodiment of the invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

In FIG. 1, an examining arrangement of a first preferred embodiment ofthe invention in the form of a fluorescence microscope for examining aspecimen 1 includes a microscope part 2, a multispectral illuminatingdevice 3 according to a first preferred embodiment of the invention anda control and evaluation unit 4 for driving the illuminating device 3and detecting and evaluating images, more precisely fluorescence images,of the specimen 1 generated by the microscope part 2.

The illuminating device 3 supplies illuminating radiation into anilluminating beam path B of the microscope part 2. The microscope part 2is configured of a collimating optic 5 for collimating the opticalradiation outputted by the illuminating device 3, a field diaphragm 6mounted downstream of the illuminating device 3, a partially transparentdeflecting mirror 7, an illuminating tube 8 having an optic, a filtercube 9 having a multiband color splitter 10 and an objective 11.Illuminating radiation exiting from the illuminating beam path thenreaches the specimen 1.

The specimen 1 is imaged by the microscope part 2. The microscope part 2has for this purpose the objective 11, the filter cube 9 and a detectiontube 12 having a detection optic which together form an imaging ordetection beam path and image the specimen 1 onto a camera 13 withdiaphragm 14. The filter cube 9 has in the imaging beam path amulticolor filter or a multiband color filter 35, which is transmissivefor the fluorescence radiation to be detected, but filters out otherspectral components, especially excitation radiation. The camera 13 isconnected via a signal connection to the control and evaluation unit 4so that images, which are detected by the camera 13 can be detected bythe control and evaluation unit 4.

For focusing the image, the microscope part 2 has an autofocusing device15 having a white light-emitting diode 15′ and an autofocus sensor 15″having a diaphragm. The autofocus device drives a z-drive (notexplicitly shown in FIG. 1) for the objective 11 by means of which theobjective 11 is displaceable for focusing along the optical axis of theobjective 11, that is, in the z-direction. The precise configuration andprecise function of the autofocus sensor 15″ are described in UnitedStates patent application publication US 2007/0102620 whose content isherewith incorporated into the disclosure by reference.

The camera 13 includes a CCD-field for receiving detection radiation andfor generating signals which reproduce the energy and therewith graysteps. The energy is received from an element of the CCD-field.

A motor-driven mechanical stage 16 serves for selecting a region of thespecimen 1 to be imaged. The mechanical stage 16 is indicated only bytwo double arrows in FIG. 1. By means of the mechanical stage 16, thespecimen 1 is movable in an xy-plane orthogonally to the z-axis. Thedrive (not shown in FIG. 1) of the mechanical stage 16 is connected viaa control connection (likewise not shown) to the control and evaluationunit 4 and is controllable thereby.

In the example, the control and evaluation unit 4 includes a personalcomputer with conventional input and output apparatus, especially amonitor not shown in FIG. 1. The illuminating device 3 and themicroscope part 2 are driveable via a graphical user interface of thecontrol and evaluation unit 4.

A monitor detector 17 is likewise connected to the control andevaluation unit 4. The monitor detector 17 is mounted behind thepartially transparent deflecting mirror 7 in the extension of theoptical axis of the collimating optic 5. The monitor detector 17receives a part of the illuminating radiation outputted by theilluminating device 3 and generates signals which reproduce the energyor intensity of the illuminating radiation. This portion of theilluminating radiation is determined by the transmission of thedeflecting mirror 7. The control and evaluation unit 4 receives thesignals and determines the intensity impinging on the specimen 1 fromthese signals.

The multispectral illuminating device 3 functions to emit opticalradiation in one or several pregiven wavelength ranges corresponding todrive signals from the control and evaluation unit 4.

For this purpose, and in addition to the white light-emitting diode 15′,the multispectral illuminating device 3 has several semiconductorradiation sources 18, in the example, four light emitting diodes (18.1,18.2, 18.3, 18.4) having respectively different emission wavelengthranges, in the example, red, green, blue or ultraviolet. Theilluminating device 3 further includes a control unit 19 which isconnected to the light emitting diodes for driving or supplying the samewith current. The following are mounted downstream of the light diodes(18.1, 18.2, 18.3, 18.4) in corresponding beam path sections: respectivecollimating units (20.1, 20.2, 20.3, 20.4); excitation band filters(21.1., 21.2, 21.3, 21.4) as well as respective color splitters (22.1,22.2, 22.3) and a mirror 23. The color splitters (22.1, 22.2, 22.3)couple the radiation of the semiconductor radiation sources (18.1, 18.2,18.3) successively into a beam path from the semiconductor radiationsource 18.4 and therewith finally into a common illuminating beam pathsection 24.

In the further course of the common illuminating beam path section 24,the following are provided: a partially transparent deflecting mirror32, an optic 25 and a homogenizing unit 26. The partially transmittingdeflecting mirror 32 functions only for the deflection and thecoupling-in of the radiation of the light emitting diode 15′ of theautofocusing unit 15 into the common illuminating beam path section 24.The homogenizing unit 26 is in the form of a mirrored hollow rod intowhich the optic 25 couples the light from the common illuminating beampath section 24. The homogenizing unit 26 functions to evenly distributethe intensity of the illuminating radiation in the illuminating beampath section 24 over the beam cross section of the illuminatingradiation so that an essentially constant intensity is achieved over thebeam cross section.

The light emitting diodes 18 each have a carrier and a surface emittingoptical radiation. In FIG. 12 a, the carrier 27 and the actual lightemitting diode chip 28 are shown by way of example for the lightemitting diode 18.1. The light emitting diode chip 28 has the radiationemitting surface 28′.

The control unit 19 is so configured that it responds to control signalsof the control and evaluation unit 4 to, on the one hand, switch on andswitch off the semiconductor radiation sources 18 individually or incombination and, on the other hand, individually controls or adjusts thebrightness of the particular switched-on semiconductor radiation sourceor the intensity of the optical radiation outputted by this source byadjusting the supply voltage. The current supply, which is provided foreach of the semiconductor radiation sources, has preferably switchingtimes in the submillisecond range, that is, faster than approximately 1ms so that a rapid switching on and switching off of each of thesemiconductor radiation sources is possible.

The collimating units 20.1 to 20.4 are arranged in the beam path ofcorresponding ones of the semiconductor radiation sources 18.1 to 18.4.The collimating units 20.1 to 20.4 collimate the optical radiationoutputted by the semiconductor radiation sources. In the example,aspheric lenses 29 of high numerical aperture, preferably greater than0.5, are used as collimating units, as shown in FIG. 12 a. Sphericallenses could, in principle, also be used, but the aspheric lenses 29have better collimating characteristics. The collimating units 20 andthe optic 25 are so designed and arranged that a component as large aspossible is coupled into the homogenizing unit 26 from each of thesemiconductor radiation sources. Especially, the collimating units andthe optic 25 are so designed in the common illuminating beam pathsection 24 forward of the homogenizing unit 26 that thelight-conductance values are optimally adapted to each other. The optic,which is formed by the collimating unit and the in-coupling optic 25 forthe homogenizing unit 26, is telecentrically designed so that the pupilof the collimating unit 20 and the pupil of the in-coupling optic 25 aresuperposed.

The optic 25 has a focal length corresponding to the necessary imagingscale and focuses the optical radiation for this purpose into the inputsurface of the homogenizing unit 26. The distances of the semiconductorradiation sources 18 to the collimating unit 26 are in each casedifferent in the present example. For this reason, the collimating units(in the example, the aspheric lenses 29) are specifically adapted inconfiguration and/or arrangement for each of the semiconductor radiationsources 18. In this example, like aspheric lenses are used whoseposition between the particular light emitting diode chip and the colorsplitter or mirror is correspondingly optimized. The color splitter ormirror follows the light emitting diode in the beam path. In analternate embodiment, specifically adapted aspherical lenses can be usedas collimating units for each of the semiconductor radiation sources incorrespondence to their position in the beam path.

As already mentioned, the semiconductor radiation sources (18.1, 18.2,18.3, 18.4) emit optical radiation with respectively different emissionspectra which are shown schematically in FIG. 2. In FIG. 2, in the samediagram, the following are shown qualitatively as a function of thewavelength λ: the emission radiation power L of the semiconductorradiation sources 18 (solid line); the transmission T_(A) of theexcitation band filters 21.1 to 21.4 (broken lines); the transmissionT_(F) of the multicolor filter 35 (dotted line); and, the transmissionT_(FT) of the color splitter 22.1. The respective emission spectra 30.1to 30.4 of the semiconductor radiation sources 18.1 to 18.4 each have apeak. The characteristic emission wavelength of the particular emissionspectrum is in this embodiment, as in the following embodiments, thewavelength having the maximum emission intensity. In another variation,the centroid wavelength (that is, the mean value of the emissionwavelengths weighted by the emission intensity) can be used ascharacteristic emission wavelength. The luminescent diodes 18.1 to 18.4therefore emit, expressed by the color of the characteristic emissionwavelength, optical radiation in spectra having decreasingcharacteristic wavelength: the light emitting diode 18.1 in red, thelight emitting diode 18.2 in green, the light emitting diode 18.3 inblue and the light emitting diode 18.4 in ultraviolet. The lightemitting diodes are so selected that the emission spectra are wellsuited for exciting at least four pregiven fluorescence colorantsdistributed over the optical spectrum from UV up to red light.

The excitation band filters 21.1 to 21.4, which are configured asinterference filters, having transmission bands 31.1 to 31.4 narrow thespectral width of the emission spectra 30.1 to 30.4 and so prevent across talk for different simultaneous fluorescence examinations fordifferent fluorescence colorants. The transmission bands 31.1 to 31.4are adapted to the respective emission spectra of the semiconductorradiation sources.

While the mirror 23 functions only for the deflection of the radiation,which is outputted by the light emitting diode 18.4, and therefore onlyhas a highest possible reflection capacity in the region of the emissionspectrum of the light emitting diode 18.4, the color splitters 22.1 to22.3 have a transmission selected in correspondence to the arrangementin the beam path and the arrangement of the light emitting diodes. Thecolor splitters 22.1 to 22.3 are assigned to corresponding ones of thesemiconductor radiation sources 18.1 to 18.3. The color splitters 22.1to 22.3 have a step-shaped transmission capacity in the wavelength rangecovered by the emission spectra of the light emitting diodes 18.1 to18.4. With this transmission capacity, the transmission for wavelengthsbelow a flank wavelength λ_(FT) is very high, whereas, above the flankwavelength λ_(FT), the reflection is very high, ideally almost 100%. Theflank wavelength λ_(FT) shows the position of the step. This form isshown schematically in FIG. 2 for the color splitter 22.1.

Along the beam path from the mirror 23 or the semiconductor radiationsource 18.4 to the homogenizing unit 26, the other semiconductorradiation sources 18.1 to 18.3 are therefore arranged in a sequence ofdecreasing characteristic emission wavelengths and the color splitters22.1 to 22.3, which are assigned thereto, are arranged in acorresponding sequence of monotone decreasing flank wavelengths.

The flank wavelengths are so selected that optical radiation from asemiconductor radiation source, which is assigned to the correspondingcolor splitter, is reflected and is thereby coupled into the beam path33 from the semiconductor radiation source 18.4 with the lowestcharacteristic emission wavelength to the homogenizing unit 26, butpasses the radiation of the other light emitting diodes whose radiationis already coupled into the beam path 33 in the direction of thesemiconductor radiation source of greatest characteristic emissionwavelength. The flank wavelengths are especially so selected that theylie in the center between the characteristic emission wavelengths of thesemiconductor radiation sources 18.

The optical excitation radiation, which is emitted by the illuminatingdevice 3, is coupled into the illuminating tube 8 by the deflectingmirror 7 after passing the collimating optic 5 and the field diaphragm6. Excitation radiation exiting from the illuminating tube 8 thenreaches the filter cube 9 which includes a multiband excitation filter34 ahead of the multiband color splitter 10. The multiband excitationfilter 34 filters out components in unwanted wavelength ranges which arepossibly still contained in the excitation radiation. In a variation ofthe embodiment, and when utilizing a so-called Pinkel filter set, thefilter 34 is not needed. A Pinkel filter set is a filter set, which issuggested by Professor Dan Pinkel, which, for example, can be used forFISH microscopy (fluorescence-in-situ-hybridizing microscopy).

The multiband color splitter 10 is at least partially reflective for theexcitation radiation. After deflection by the multiband color splitter10, the excitation radiation is focused on the specimen 1 by theobjective 11 and there excites fluorescence radiation characteristic forthe particular fluorescence colorant when corresponding fluorescencecolorants are present.

The detection radiation emanating from the specimen 1, which especiallyincludes the excited fluorescence radiation, is parallelized by theobjective 11 and reaches the multiband color splitter 10 which is sodesigned that optical radiation in the wavelength ranges, in which thefluorescence radiation of the given fluorescence colorants lies, ispassed but that other radiation is deflected. The passed detectionradiation is then again filtered by the multicolor filter or emissionfilter 35, which is transmissive in narrow wavelength ranges about thewavelengths of the expected fluorescence radiation emanating from thepregiven fluorescence colorants, but not for radiation havingwavelengths between these ranges. The transmission of one suchmulticolor filter 35, which is matched to the illuminating device 3, isexemplary and shown schematically in FIG. 2 by a dotted line.

The remaining radiation is imaged by the detection optic in the detectortube 12 onto the camera 13 having diaphragm 14 where the occurring imageis detected. The occurring detection signals are then detected by thecontrol and evaluation unit 4 whereby, overall, an image of thedistribution and concentration of the pregiven colorant substances isdetected in the specimen 1.

To generate the images, the semiconductor radiation sources 18 can beswitched on sequentially or in combination. The intensity of theparticular outputted optical radiation can be controlled by the controlunit 19. This is especially advantageous when an ocular is used for thedirect viewing in lieu of the camera 13.

The following four preferred embodiments of an examining arrangementaccording to the invention differ from the examining arrangement of thefirst embodiment by the configuration of the illuminating device 3. Inlieu of the illuminating device 3, other preferred embodiments ofilluminating devices according to the invention are used. Theautofocusing unit is dispensed with. All other parts are unchangedexcept for matching to the changed illuminating device so that thedescription with respect to the first embodiment also applies here.Also, in the illuminating device, for some components, which correspondto the first embodiment, the same reference characters are used.

In the FIGS., the control unit for the semiconductor radiation sourcesis not shown in each case. The control unit is different from the firstembodiment only in that the control unit is configured for driving fivesemiconductor radiation sources. The drive of the individualsemiconductor radiation sources can be as in the first embodiment.

What was said for the first embodiment with respect to the configurationand arrangement of the collimating units 20, the in-coupling optic 25and the homogenizing unit also applies here.

The illuminating devices each have five semiconductor radiation sourcesin lieu of four semiconductor radiation sources and the characteristicemission wavelengths of the emission spectra are different and aredistributed over the spectral range from the UV to the red end of thevisible spectrum. Especially one of the semiconductor radiation sourcesis a radiation source for optical radiation in the UV range.

The semiconductor radiation sources do not differ between the followingembodiments. Accordingly, the semiconductor radiation sources areidentified by the same reference numeral 36. The radiation sources canbe selected in the form of light emitting diodes as shown below. If thenumerals 36.1 to 36.5 identify the respective semiconductor radiationsources in one embodiment, then, for example, the light emitting diode36.1 can emit radiation having a characteristic emission wavelength inthe IR or red region of the optical spectrum; light emitting diode 36.2can emit radiation having a characteristic emission wavelength in theyellow region of the spectrum; light emitting diode 36.3 can emitradiation having a characteristic emission wavelength in the greenregion of the spectrum; light emitting diode 36.4 can emit radiationhaving a characteristic emission wavelength in the blue region of thespectrum; and, light emitting diode 36.5 can emit radiation having acharacteristic emission wavelength in the UV range.

In the first of four embodiments, which is shown schematically in FIG.3, the optical radiations of the semiconductor radiation sources 36.1 to36.5 are, in the direction from the radiation source 36.1 to thehomogenizing unit 26, successively coupled by means of fourcorresponding color splitters 37.1 to 37.4 into a beam path (which islinear in the example) from the radiation source 36.1 to downstream ofthe color splitter 37.4. The end of the beam is defined by the commonilluminating beam path section 24.

As in the first embodiment, the four color splitters 37.1 to 37.4 have astep-shaped transmission in the optical wavelength range. This is shownin FIG. 4 which shows the respective transmission for each of the fourcolor splitters as a function of the wavelength λ as a solid line andshows the emission spectra of the light emitting diodes 36.1 to 36.5,that is, the intensity as a function of the wavelength as broken linesor dotted lines.

While the radiation of the light emitting diode 36.1 is not deflected,the light emitting diodes 36.2 to 36.5 are arranged sequentially alongthe beam path of the light emitting diode 36.1 in a sequence ofdecreasing wavelength. The color splitters 37.1 to 37.4 are arranged at45° relative to the linear beam path emanating from the light emittingdiode 36.1 in order to couple the radiation of the light emitting diodes36.2 to 36.5 into the common illuminating beam path section 24.

Here too, respective collimating units 20.1 to 20.5 are mounted betweenthe light emitting diodes and the color splitters 37.1 to 37.4, whichlie closest in the beam path, and excitation filters 21.1 to 21.5 aremounted between the collimating units and the color splitters. Theexcitation filters 21.1 to 21.5 are configured as in the firstembodiment except for the matching to the centroid wavelengths of thelight emitting diodes 36.1 to 36.5.

The radiation from the common illuminating beam path section 24 reachesdirectly into the optic 25, which is configured in the same manner as inthe first embodiment, and directs the illuminating beam onto the entrysurface of the homogenizing unit 26 which is likewise configured as inthe first embodiment.

In FIG. 4, the emission spectra of the light emitting diodes are shownfor each of the color splitters by dotted lines and the transmission ofthe particular color splitter is shown as a solid line marked with thereference numeral of the color splitter. As shown in FIG. 4, the filteredges or filter flanks 38.1 to 38.4 of the color splitters are arrangedmonotonically decreasing along the beam path emanating from the lightemitting diode 36.1 in the illuminating direction as a function of thewavelength in the same manner as the wavelength from the radiationsource 36.1 to the homogenizing unit 26 of the light emitting diode 36so that radiation of the previous light emitting diodes is passed butradiation of the next light emitting diode is deflected and is coupledinto the common beam path. In the ideal case, the transmission of thecolor splitter in the intended pass region is 100% (T=1) and thereflectivity at 45° in the same region is 0% (R=0) and vice versa. Thatis: T+R=1.

The multiband filter of the filter cube 9 and the multiband colorsplitter 35 are configured in the same manner as in the first embodimentbut here for the optical radiation of the five light emitting diodes.

This second embodiment is especially advantageous when the radiation ofthe semiconductor radiation source having the lowest characteristicemission wavelength (that is, in the example, light emitting diode 36.5)should be coupled into the homogenizing unit 26 with especially highefficiency. The reason for this is the short distance of thissemiconductor radiation source to the homogenizing unit 26. Since highpower light emitting diodes output the radiation with a large anglespectrum, the radiation itself can be only approximately collimated(parallelized) even with a well adapted aspheric lens. For this reason,the losses are dependent clearly on the path length which the collimatedradiation must traverse.

The light emitting diodes can also be arranged in the reverse sequence,that is, in a sequence of monotone increasing centroid wavelength sothat the light emitting diode 36.5 has the longest wavelength. In thiscase, the color splitters are to be inverted with respect to theirtransmission capacity, that is, transmission range and reflection rangeare exchanged. This means that the color splitters are arranged in asequence of monotone increasing flank wavelengths along the illuminatingdirection.

FIG. 5 shows a third embodiment which differs from the previousembodiment only by the arrangement of the semiconductor radiationsources having the collimating units and the excitation filters as wellas the color splitters. The arrangement of semiconductor radiationsource and the collimating unit corresponding thereto, the assignedexcitation filter and the color splitter is essentially unchanged withrespect to the previous embodiment except for, as required, thedistances from the collimating unit to the semiconductor radiationsource. Accordingly, the same reference numerals as in the previousembodiment are used. Especially, the transmission characteristics of thecolor splitters 37 compared to the previous embodiment are unchanged.

In this embodiment, the semiconductor radiation sources and the colorsplitters are so arranged that all semiconductor radiation sources areat the same distance to the last color splitter 37.4.

For this purpose, the beam paths from the semiconductor radiationsources to the common illuminating beam path section 24 are soconfigured that the beam path sections of two semiconductor radiationsources 36.3 and 36.4 are coupled into a common beam path section viathe color splitter 37.3 which, in turn, is coupled by means of colorsplitter 37.2 with a beam path section leading from the semiconductorradiation source 36.1 without deflection to the common illuminating beampath section 24. The radiation of the remaining two semiconductorradiation sources 36.2 and 36.5 is coupled into the common illuminatingbeam section 24 via corresponding color splitters 37.1 and 37.4 forwardand rearward of color splitter 37.2, respectively.

The transmission of the color splitters is shown in FIG. 6 which isanalog to FIG. 4. Here, for each of the color splitters, the emissionspectra of the light emitting diodes are shown by dotted lines and thetransmission of the particular color splitter is shown by a solid linemarked with the reference numeral of the color splitter. The flankwavelengths of the color splitters having step characteristics lie, ineach case, below the emission range of the assigned light emitting diodebut above the emission ranges of the light emitting diodes havingemission ranges of shorter wavelengths.

As in the previous embodiments, respective excitation filters aremounted between corresponding ones of the collimating units, whichfollow the semiconductor radiation sources 36 in the beam path, and thecolor splitters closest in the beam path.

This embodiment satisfies basically the same function as the previousembodiment. The arrangement is, however, so selected that allsemiconductor radiation sources have the same distance to the last colorsplitter or to the homogenizing unit 26. In this way, all semiconductorradiation sources can be treated in the same way with respect to thecollimating optic 5 which facilitates the practical application. If thesemiconductor radiation source 36.5 is moved closer to the colorsplitter 37.4, then the semiconductor radiation sources 36 have onaverage a very short distance to the homogenizing unit 26 whichincreases efficiency and contributes to a compact configuration.

FIG. 7 shows a fourth embodiment for the illuminating device in theexamining arrangement according to the invention which differs from theprevious embodiment only in the arrangement of the semiconductorradiation sources 36 and the color splitters 37. Furthermore, the colorsplitter 37.4 is replaced by a color splitter 37.4′. Accordingly, forthe same elements, the same reference characters are used and thedescriptions as to these elements for the previous embodiment apply hereas well. As in the previous embodiments, the excitation filters arearranged between the collimating units 20, which follow the respectivesemiconductor radiation sources 36 in the corresponding beam paths andthe respective color splitters 37, which are next in the beam path.Furthermore, the semiconductor radiation sources and the color splittersare also so arranged that all semiconductor radiation sources are at thesame distance to the last color splitter 37.4′.

In this embodiment, the color splitters 37 form knots in a tree whosebranches are radiation path sections from the semiconductor radiationsources 36 to the color splitters 37 and/or between the color splitters37. The beam path of the semiconductor radiation source 36.5 isessentially linear.

The transmission of the color splitters is shown in FIG. 8 which is thesame as in FIG. 4. In FIG. 8, the emission spectra of the light emittingdiodes are shown for each color splitter by a dotted line and thetransmission of the respective color splitter is shown by a solid linemarked with the reference character of the color splitter. As shown inFIG. 8, the color splitter 37.4′ has the inverse function compared tocolor splitter 37.4 and is used here in transmission for thesemiconductor radiation source 36.5. This variation shows by way ofexample the variation possibilities for the selection of the colorsplitters.

The beam paths of the other semiconductor radiation sources (36.1, 36.2)and (36.3, 36.4) are coupled via color splitters 37.1 and 37.2,respectively, with the first beam path sections which, in turn, arecoupled by means of color splitter 37.3 and directed to color splitter37.4.

FIG. 9 shows a fourth preferred embodiment of the illuminating deviceand therewith of the imaging unit of the invention and differs from theprevious embodiment by the arrangement of the semiconductor radiationsources and the color splitters corresponding thereto as well as thecharacteristic of some of the color splitters. Therefore, for allcomponents, the same reference characters will be used as in the firstor previous embodiment.

In contrast to the previous embodiment, one of the semiconductorradiation sources 36 (in the example, the semiconductor radiation source36.3) is so arranged that it radiates through only one of the colorsplitters 37, namely, the color splitter 37.3′, before its radiation iscoupled into the common illuminating beam path section. The emissionspectrum of this one semiconductor radiation source 36 lies with itscharacteristic emission wavelength between at least two emission spectraor the centroid wavelengths of these emission spectra. The foregoing isespecially advantageous when the semiconductor radiation source isespecially weak in power in comparison to the others. According to thepresent state of the art, this is the case, for example, for green lightemitting diodes. Additionally, the possibility is present here toarrange this semiconductor radiation source closer to the color splitterso that it can be coupled into the homogenizing unit 26 with maximumefficiency.

The transmission of the color splitters is shown in FIG. 10 which isanalog to FIG. 4. In FIG. 10, the emission spectra of the light emittingdiodes are shown for each of the color splitters by dotted lines and thetransmission of the particular color splitter is shown as a solid linewith the line marked with the reference character of the color splitter.In contrast to the previous embodiment, the color splitter 37.3′ has nostep characteristic and instead operates as a bandpass for the opticalradiation of the light emitting diode 36.3 assigned to this colorsplitter. The other color splitters are, except for the color splitter37.4 which corresponds to that of the penultimate embodiment, unchangedrelative to the previous embodiment.

Further preferred embodiments can be varied by: change of the number ofsemiconductor radiation sources and the number of color splitters,semiconductor radiation sources or adding semiconductor radiationsources with color splitters adapted thereto.

As a further embodiment, FIG. 11 shows an illuminating arrangementhaving 2^(N) semiconductor radiation sources 39.1 to 39.8 withrespectively different emission spectra and 2^(N)-1 color splitters 40.1to 40.7 for N=3 wherein the semiconductor radiation sources 39 arearranged at the ends of the branches of a binary tree and thereby defineleaves of the tree. The branches or knots of the tree are formed by thecolor splitters 40.1 to 40.7 and the transmission regions of these colorsplitters are spectrally arranged in the same manner as in the previousembodiments. This illuminating device is very advantageous with respectto the optimization of the energy efficiency because the illuminatingdevice is configured to be very compact. The maximum packing density ofsemiconductor radiation sources can be obtained when the color splittersare arranged in a tree having 2^(N) side arms. Here too, respectivecollimating units 20.1 to 20.8 and excitation filters 21.1 to 21.8 arearranged between the semiconductor radiation sources and the colorsplitters. The excitation filters 21.1 to 21.8 are configured asinterference filters and are configured in the same manner as in thesecond embodiment.

There are still two optional additional collimating optics 42.1 and 42.2provided ahead of the last color splitter 40.4 which effect a stillbetter collimation.

Even though the semiconductor radiation sources and the color splittersare arranged in a plane in the embodiments, this need not necessarily bethe case. Rather, these can be rotated about sections of the overallbeam path in order to achieve a best possible suitable form of theilluminating device for the application.

A further examining arrangement is shown schematically in FIG. 13 anddiffers from the examining arrangement of the first embodiment in thatit has a receptacle for at least two filter cubes (9, 9′) withexcitation filter sets and emission filter sets by means of which eachone of the filter cubes can be moved into the detection beam path of thearrangement. The filter edges of the multiband filter (35, 35′) arepreferably displaced relative to each other by a distance between 10 nmand 90 nm. The receptacle 41 (in the present example, a filter wheel) ispreferably movable (in the example, rotatable) by means of a motorizeddrive (not shown in FIG. 13) in response to corresponding signals of thecontrol and evaluation unit 4.

As a rule, more emission wavelength ranges of the illuminating deviceare available than are bands per multiband filter set. For this reason,a high flexibility can be obtained with the use of the filter wheel.

A further preferred embodiment of an illuminating device differs fromthe illuminating device of FIG. 1 in that operator-controlled elements,for example, switches, are provided on a housing of the illuminatingdevice by means of which the semiconductor radiation sources can beswitched on and off individually or in combination.

FIGS. 12 b and 12 c show two additional alternatives for the collimatingunits for otherwise unchanged illuminating devices of the previousembodiments.

In FIG. 12 b, a diffractive or holographic element 43 is provided inaddition to an aspheric lens 29 on the light emitting diode chip 28. Theelement 43 increases the radiation yield of the light emitting diode inthat it better couples the radiation into the aspheric lens 29.

In FIG. 12 c, a radiation concentrator or light concentrator 44 ofplastic or glass is mounted in the beam path ahead of the light emittingdiode in lieu of the aspheric lens 29. The concentrator 44 collimatesthe light entering the concentrator via total reflection at the boundarysurfaces of the light concentrator.

Another preferred embodiment differs from the second embodiment by theconfiguration of one of the semiconductor radiation sources. In order tobe able to make available an optical radiation in the wavelength rangeabout 570 nm, a light emitting diode is used to output blue light or alight emitting diode is used to output UV radiation. The light emittingdiode contains luminescent substances which lead to a radiation emissionin the desired spectral range. The UV light or blue light of the lightemitting diode is converted via fluorescence or phosphorescence intolight in the desired wavelength range, that is, of another color.

The above-mentioned examining arrangements can be used for determiningthe concentration of the pregiven fluorescence colorants. The filterbands and the characteristic emission wavelengths are so matched to eachother that the fluorescence excitation and fluorescence detection are asefficient as possible for the pregiven fluorescence colorant.

If the excitation spectra and fluorescence spectra are known for M (Mpositive natural number) fluorescence colorants as well as the emissionspectra of the semiconductor radiation sources and the filters, then theconcentrations of the M fluorescence colorants in the specimen 1 canbasically be determined with M spectrally different measurements. Inorder to obtain spectrally different measurements, at least oneexcitation wavelength or one fluorescence wavelength must be differentthan in the remaining measurements. From the excitation standpoint, thiscan be realized via switching on or switching off of the individualsemiconductor radiation sources. Another possibility is to exchange themultiband filter set.

Preferably, however, several excitation wavelengths are usedsimultaneously for the measurements by switching on at least two of thesemiconductor radiation sources.

The examining arrangement of the first embodiment can especially be usedfor carrying out a preferred embodiment of the method of the inventionfor measuring the concentration of a number M of pregiven fluorescencecolorants in the specimen 1. Here, M measurements are carried out. Foreach of the measurements, another number less than or equal to M ofemission wavelength ranges is used which corresponds to the excitationspectra of the fluorescence colorants for forming excitation radiation.The specimen is irradiated with the excitation radiation and theresultant fluorescence radiation is detected. The concentration of thefluorescence colorants is determined from the detection results of the Mmeasurements.

This is described for the example of a specimen with M=3 pregivenfluorescence colorants, in the example, FITC, CY3 and Cy5.

For this purpose, a 3-band filter set is used, for example, the filterset 61005 of the Chroma Technology Company. With each additionalmeasurement, a further emission band is used in addition to the emissionbands used in the previous measurement, that is, successively moreemission bands are used for the measurements. Accordingly, in the firstmeasurement, only one semiconductor radiation source (in the example,the blue light emitting diode) is switched on. In the secondmeasurement, an additional semiconductor source is switched on so thatthe blue and the green light emitting diodes are used. In the thirdmeasurement, a still further emission spectrum is included by switchingon the third semiconductor radiation source (in the example, the redlight emitting diode) so that all three light emitting diodes operateand emission radiation or excitation radiations in the blue, green andred range are simultaneously available.

The three measurements supply a linear equation system withconcentrations for the three fluorescence colorants as unknownvariables. The equation system is tri-diagonal and can clearly be solvedwith known mathematical methods (see, for example, William H. Press,Saul A. Teukolsky, William T. Vetterling: “Numerical Receipes in C”, 2ndedition, Cambridge University Press 1999, ISBN: 0521431085) and theconcentration ratios can thereby be determined. Cross talk effects areeliminated which could occur when using several excitation bands.

The accuracy of the concentration determination can be increased withfurther measurements, especially, with other excitation wavelengths.

For this purpose, especially a filter set can be used wherein all filteredges are shifted by several 10 nm to higher or lower wavelengthsrelative to the first filter set. In this way, other regions of theexcitation spectra and emission spectra of the fluorescence colorantsare utilized which supply additional information.

The advantages of the illuminating device of the invention explained formicroscopy can also be used for other applications. For example, theilluminating device can be used as illumination for digital projectionsystems when an especially extensive color space is to be covered. A usefor general illuminating purposes is likewise conceivable when a rapidvariability of the color spectrum is a consideration. Especially,optical devices for examining the eye or devices for examining human oranimal tissue, for example, surgical microscopes or arrangements forexamining the eye such as fundus cameras, can be equipped with anilluminating device according to one of the above embodiments.

The illuminating device permits a color true illustration by means ofcolor management insofar as a viewing or detection in reflection takesplace. Color management is understood to mean especially that in thearrangement, the illuminating device and an image detecting unit fordetecting images of an object, which arise under illumination by theilluminating device, the spectral characteristics of the illuminatingdevice and the image detection unit are so matched to each other thatfor a human viewer, the same colors result as in the viewing with theeye under a standard illumination, for example, a halogen lamp having apregiven color temperature, for example, of 3200 K.

The invention claimed is:
 1. An optical arrangement configured toexamine a specimen comprising: an optical assembly defining an opticalaxis; an illuminating device for supplying optical radiation along anilluminating beam path; means for coupling said optical radiation intosaid optical assembly for transmission along said optical axis; and,said illuminating device including: at least two light emitting diodesfor emitting optical radiation with respectively different emissionspectra and along respective beam path segments; said means for couplingincluding at least one color splitter assigned to each of said lightemitting diodes, respectively; each of said color splitters beingreflective for the optical radiation of the light emitting diodecorresponding thereto; said light emitting diodes and said colorsplitters being so arranged that the optical radiation emitted by eachof said light emitting diodes is coupled into a common illuminating beampath segment; a plurality of collimating units mounted in said beam pathsegments, respectively, to collimate the optical radiation emitted bycorresponding ones of said light emitting diodes and passing torespective ones of said color splitters; said optical arrangement beingconfigured to conduct fluorescence examinations of said specimen; saidoptical assembly defining a detection beam path along which fluorescenceradiation emanating from said specimen travels; said detection beam pathfor said fluorescence radiation including at least one multibandemission filter or multiband emission filter set; wherein at least oneof said emission spectra of said light emitting diodes lies with itscharacteristic wavelength between two emission wavelength ranges of saidmultiband emission filter or said multiband emission filter set; and,said optical assembly further comprising a white-light emitting diodebeing coupled into said common illuminating beam path by a partiallytransmitting deflecting mirror; wherein the optical radiation emitted byeach of said light emitting diodes is reflected by said partiallytransmitting deflecting mirror and the optical radiation emitted by saidwhite-light emitting diode is transmitted through said partiallytransmitting deflecting mirror.
 2. The optical arrangement of claim 1,wherein the collimating units are aspheres.
 3. The optical arrangementof claim 2, wherein positions of the collimating units are adjustablerelative to the respective light emitting diodes.
 4. The opticalarrangement of claim 1, wherein said optical arrangement is configuredas a wide field microscope or a fluorescence reader.
 5. The opticalarrangement of claim 1, wherein said multiband emission filter iscontained in a filter cube and, wherein said optical arrangement furtherincludes a multiband excitation filter or multiband excitation filterset and a multiband color splitter for deflecting said optical radiationof said illuminating device or said fluorescence radiation emanatingfrom said specimen; said multiband excitation filter or said multibandexcitation filter set being mounted in said illuminating beam path ofsaid illuminating device.
 6. The optical arrangement of claim 5, whereinsaid multiband excitation filter is a first multiband excitation filterand said optical arrangement further includes a second multibandexcitation filter; and, said multiband excitation filters have filteredges shifted relative to each other by a distance of between 10 nm and90 nm.
 7. The optical arrangement of claim 1, further comprising areceptacle for at leapt two filter cubes by means of which one of thefilter cubes is movable into said detection beam path.